There has been increasing interest in the syntheses of carbohydrates due to their profound role in various biological processes and thus their potential in drug development. Various methods have been developed for the preparation of these oligosaccharides from simpler carbohydrate derivatives both chemically and enzymatically but they must be carefully applied to obtain the desired linkages. The main method that has been used to control the regioselectivity of glycosylations is protecting group chemistry. Unfortunately this requires additional steps lengthening the synthesis sequence. Extensive literature has shown the ability for carbohydrate recognition via organoboron methods through selective binding and their ability to undergo regioselective glycosylation through sugar-derived boronate esters. Exploiting these factors and in extension of previous work done in our laboratory on borinic acid- and boronic acid-catalyzed regioselective glycosylations of carbohydrate derivatives, we proposed a target oriented synthesis of a pentassacharide moiety found in four saponins isolated from Spergularia ramosa.

There has been increasing interest in the syntheses of carbohydrates due to their profound role in various biological processes and thus their potential in drug development. Various methods have been developed for the preparation of these oligosaccharides from simpler carbohydrate derivatives both chemically and enzymatically but they must be carefully applied to obtain the desired linkages. The main method that has been used to control the regioselectivity of glycosylations is protecting group chemistry. Unfortunately this requires additional steps lengthening the synthesis sequence. Extensive literature has shown the ability for carbohydrate recognition via organoboron methods through selective binding and their ability to undergo regioselective glycosylation through sugar-derived boronate esters. Exploiting these factors and in extension of previous work done in our laboratory on borinic acid- and boronic acid-catalyzed regioselective glycosylations of carbohydrate derivatives, we proposed a target oriented synthesis of a pentassacharide moiety found in four saponins isolated from Spergularia ramosa.

Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-24T15:56:48Z No. of bitstreams: 2 Dissertação - Carla Santos de Freitas - 2011.pdf: 16568607 bytes, checksum: 4e9c69288ffc7c2fe8e140e5cf8e7ff5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-26T15:42:23Z (GMT) No. of bitstreams: 2 Dissertação - Carla Santos de Freitas - 2011.pdf: 16568607 bytes, checksum: 4e9c69288ffc7c2fe8e140e5cf8e7ff5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-26T15:42:23Z (GMT). No. of bitstreams: 2 Dissertação - Carla Santos de Freitas - 2011.pdf: 16568607 bytes, checksum: 4e9c69288ffc7c2fe8e140e5cf8e7ff5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2011-06-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Galianthe ramosa is a shrub of Rubiaceae family (tribe Spermacoceae) with occurrence on Brazilian Cerrado. Phytochemical studies of Rubiaceae shows a wide variety of secundary metabolites with large biological potential and several species are used on folk medicine or as phytotherapy. The only chemical study about this genera, G. brasiliensis describes iridoids isolation. In our continuing chemical and biological investigations of plants from the family Rubiaceae from Cerrado, herein we report the isolation of new alkaloids 1 and 2, together with the known epi-catechin, ursolic acid and oleanolic acid. Besides we propose a biogenic route for new alkaloids because they have been present a new terpene portion. Their structures were determined by IR, HRESIMS, and 1D and 2D NMR experiments, and antioxidant activities, evaluated by assaying the free radical scavenging capacity using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical as substrate. The antioxidant activities of crude extract from flowers, leaves, roots and branches showed weakly activities which EC50 105, 107, 109 and 110 μg/mL respectively. / Galianthe ramosa é um subarbusto da família Rubiaceae (tribo Spermacoceae) com ocorrência no Centro Oeste do Brasil. Estudos fitoquímicos de Rubiaceae revelaram uma grande diversidade de metabólitos secundários com alto potencial biológico fazendo com que muitas espécies dessa família sejam amplamente utilizadas na medicina popular e na obtenção de fitofármacos e fitoterápicos. O único estudo químico encontrado sobre espécies deste gênero é o da espécie G. brasiliensis, onde é descrito o isolamento de iridóides. No presente trabalho realizou-se a investigação fitoquímica de G. ramosa, que teve entre outros objetivos e a avaliação do potencial antioxidante dos extratos brutos e a contribuição à quimiotaxonomia desta família. A partir do fracionamento dos extratos brutos foi possível o isolamento de dois alcalóides indol-monoterpenos inéditos e de outros três compostos já conhecidos e isolados em outras espécies da família: epi-catequina, ácido ursólico e ácido oleanólico. Uma vez que os alcalóides isolados são constituídos por um novo esqueleto terpênico, propomos também uma rota biogênica para o mesmo. Os extratos brutos das flores, folhas, raízes e galhos reagiram com o radical DPPH levando a uma perda de 50% na absorbância em concentrações de 105, 107, 109 e 110 μg/mL respectivamente, mostrando-se pouco promissores quanto ao potencial antioxidante.

Parasites are thought to have large effects on their host populations, driving genetic change, population density changes, speciation and be a major selective force maintaining sexual reproduction. Indirect signatures of parasite-mediated selection are common, but explicit examples of parasite-mediated selection in nature are lacking. In this thesis I examine parasite-mediated dynamics in a natural population of Daphnia magna that experiences an annual epidemic of the bacterial pathogen Pasteuria ramosa. I also test a novel hypothesis investigating the relationship between parasitism and the production of resting eggs. In chapter 2 a combined field study and laboratory infection experiment illustrates one of the best examples of parasite-mediated selection in a natural population, with Daphnia collected after a parasite epidemic having higher levels of parasite resistance than those collected before. This chapter also explored the relationship between parasitism and resting eggs, which are only produced during the sexual phase of reproduction. Daphnia that were reproducing sexually in the field prior to the parasite epidemic were more susceptible, supporting higher levels of parasite growth, than their asexual counterparts. This supports the idea that some genotypes invest in sex at the expense of parasite resistance. In chapter 3 I used molecular markers to investigate genotype frequency changes in the same population in relation to the parasite epidemic. The parasite epidemic was found to be associated with genetic change in the population, and a laboratory infection experiment revealed that the genotype most resistant to the parasite was also most common following the peak of the parasite epidemic. While chapter 2 explored a genetic relationship between susceptibility and resting eggs, chapter 4 explores whether crowding conditions, cues indicating parasite prevalence in the population, or direct exposure to parasite spores can induce resting egg production. I found that crowding conditions or parasite prevalence enhance levels of male and resting egg production, but patterns were entirely dependent on Daphnia genotypes. There was no indication that exposure to parasite spores affects levels of sexual reproduction.

<i>Orobanche</i> species are parasitic weeds that subsist on the roots of many dicotyledonous plants. These parasites form symplastic and apoplastic connections with their hosts and act as strong sinks for the uptake of water, minerals, and photosynthates, often causing severe damage to the hosts. Although the uptake of small molecules such as sugars and herbicides by <i>Orobanche</i> has been documented, movement of macromolecules between host and parasite has not been characterized. The objectives of this research were to 1) determine whether, and by what route, host macromolecules can be translocated to the parasite, and 2) engineer host resistance based on inducible expression of sarcotoxin IA, an anti-microbial peptide from the flesh fly (<i>Sarcophaga peregrina</i>). To address the first objective, transgenic plants expressing GFP localized to either the host cell cytosol (symplast) or secreted to the extra-cellular space (apoplast) were parasitized by <i>O. aegyptiaca</i>. Observations of green fluorescence in <i>O. aegyptiaca</i> tubercles growing on these plants indicate that the 27 kDa GFP molecule was translocated to the parasite via both symplastic and apoplastic routes. This work was supported by studies with xylem- and phloem-specific dyes, which showed that fluorescent dextrans as large as 70 kDa moved into the parasite through xylem connections. The second objective was addressed using tobacco (<i>Nicotiana tabacum</i> L. cv. Xanthi) plants expressing the sarcotoxin IA transgene under control of the parasite-inducible <i>HMG2</i> promoter. In soil experiments, transgenic tobacco plants had greater height and biomass, and showed up to 90% reduction in <i>O. ramosa</i> parasitism as measured by the fresh weight of parasite tubercles. In a semi-hydroponic growth system, where <i>Orobanche</i> tubercles can be visualized at early stages of growth, <i>O. aegyptiaca</i> parasites growing on plants expressing sarcotoxin IA were smaller and had an increased number of senescent tubercles compared to those growing on non-transformed plants. Considering the relatively small size of sarcotoxin IA (4 kDa), it is likely that this peptide moves from host to the parasite, where it accumulates to phytotoxic concentrations. In addition to increasing our knowledge of host-<i>Orobanche</i> interactions, this research used an antibiotic peptide to engineer partial <i>Orobanche</i> resistance into a highly susceptible crop. This strategy has broad implications for the control of other parasitic weeds. / Ph. D.

Made available in DSpace on 2016-06-02T19:29:57Z (GMT). No. of bitstreams: 1 5307.pdf: 3018065 bytes, checksum: 4a4ed79b44dafeeb1204798dc8f70256 (MD5) Previous issue date: 2013-06-03 / Universidade Federal de Sao Carlos / This thesis aimed to evaluate the use of cholinesterases (ChE) of the cladoceran species Pseudosida ramosa and Daphnia magna as a biochemical biomarker of the presence and effects of anatoxin-a(s) at different levels of biological organization (molecular, individual and population), besides the combined effects of the mixtures of the hepatotoxic (microcystins) and neurotoxic (anatoxin-a(s)) extracts in D. magna. A microplate assay was adapted and optimized for measuring the ChE activity of P. ramosa, in order to produce an assay protocol for this species. The analysis on the performance of ChE assays in P. ramosa showed that these are suitable for the quantifying of enzymatic activity in this species. P. ramosa showed to be an adequate alternative to the exotic cladoceran D. magna. Thus, it was proposed an assay protocol, which it meets the best combination of parameters for the using of ChE activity of P. ramosa as a biochemical biomarker. The ChE activity of P. ramosa and D. magna were specific for the indication of the presence of anatoxin-a(s), since no effect on the enzymatic activity of these species was observed when they were exposed to the microcystins. In the acute exposures (48-h) to the anatoxin-a(s) extract and to the paraoxon-methyl, P. ramosa was more sensitive than D. magna for ChE activity and survival endpoints. Also, P. ramosa was more sensitive than D. magna when exposed to the anatoxin-a(s) extract for 7 days. When the relationships between the ChE inhibition and individual and populational endpoints were evaluated, different responses were observed for the studied species. The ChE inhibition in P. ramosa had a very close relationship with the survival in the acute exposures to the anatoxin-a(s) extract and to the paraoxon-methyl. For D. magna, on the other hand, this relationship was not linear, being high levels of ChE inhibition associated with almost no mortality. The ChE activity in P. ramosa was also a good predictor of the chronic effects of anatoxin-a(s) extract at higher levels of biological organization, since ChE inhibition (48 h) was linearly linked to the sub-lethal effects on the reproduction (21 days) and on the population growth rate (21 days). For D. magna, these relationships could not be established, possibly due to species-specific differences in the affinities of both acetylcholinesterase and pseudocholinesterases to the toxicants. Thus, for the using of ChE as a biochemical biomarker in the risk assessments of neurotoxic cyanobacteria blooms in tropical regions, it is recommended the use of native species, especially of P. ramosa, since the model species D. magna could overestimate the risk to the local species. When the effects of the mixtures of the hepatotoxic and neurotoxic extracts were evaluated on the survival and feeding rates of D. magna, additive and synergistic responses were only observed on the feeding rates. Therefore, since different types of cyanotoxins are found in the natural environments in combination, the risks of these toxins on the zooplanktonic community should be evaluated not only individually, but also as mixtures. / Esta tese teve como objetivo avaliar o uso das colinesterases (ChE) das espécies de cladóceros Pseudosida ramosa e Daphnia magna como um biomarcador bioquímico da presença e dos efeitos de anatoxina-a(s) em diferentes níveis de organização biológica (molecular, individual e populacional), além dos efeitos combinados das misturas dos extratos hepatotóxicos (microcistinas) e neurotóxicos (anatoxina-a(s)) em D. magna. Um ensaio de microplacas foi adaptado e otimizado para medir a atividade de ChE da P. ramosa, a fim de produzir um protocolo de ensaio para esta espécie. A análise sobre o desempenho dos ensaios de ChE em P. ramosa mostrou que estes são adequados para a quantificação da atividade enzimática nesta espécie. P. ramosa mostrou ser uma alternativa adequada para o cladócero exótico D. magna. Assim, foi proposto um protocolo de ensaio, o qual reúne a melhor combinação de parâmetros para a utilização da atividade de ChE da P. ramosa como um biomarcador bioquímico. A atividade de ChE da P. ramosa e da D. magna foram específicas para a indicação da presença de anatoxinaa( s), uma vez que nenhum efeito sobre a atividade enzimática dessas espécies foi observado quando elas foram expostas às microcistinas. Nas exposições agudas (48 h) ao extrato de anatoxina-a(s) e ao paraoxon-metil, P. ramosa foi mais sensível do que D. magna para os parâmetros atividade de ChE e sobrevivência. Também, P. ramosa foi mais sensível do que D. magna quando exposta ao extrato de anatoxina-a(s) por sete dias. Quando as relações entre a inibição de ChE e os parâmetros individuais e populacionais foram avaliados, diferentes respostas foram observadas para as espécies estudadas. A inibição de ChE em P. ramosa teve uma relação muito próxima com a sobrevivência nas exposições agudas ao extrato de anatoxina-a(s) e ao paraoxon-metil. Para D. magna, por outro lado, esta relação não foi linear, sendo níveis altos de inibição de ChE associados com quase nenhuma mortalidade. A atividade de ChE em P. ramosa foi também um bom preditor dos efeitos crônicos do extrato de anatoxina-a(s) em níveis mais elevados de organização biológica, uma vez que a inibição de ChE (48 h) foi associada linearmente aos efeitos sub-letais na reprodução (21 dias) e na taxa de crescimento populacional (21 dias). Para D. magna, essas relações não puderam ser estabelecidas, possivelmente devido a diferenças espécie-específicas nas afinidades da acetilcolinesterase e das pseudocolinesterases aos tóxicos. Assim, para a utilização de ChE como um biomarcador bioquímico nas avaliações de risco de florescimentos de cianobactérias neurotóxicas em regiões tropicais, recomenda-se o uso de espécies nativas, especialmente da P. ramosa, uma vez que a espécie modelo D. magna poderia superestimar o risco para as espécies locais. Quando os efeitos das misturas dos extratos hepatotóxicos e neurotóxicos foram avaliados sobre a sobrevivência e as taxas alimentares da D. magna, respostas aditivas e sinergísticas foram observadas apenas nas taxas alimentares. Portanto, uma vez que diferentes tipos de cianotoxinas são encontrados nos ambientes naturais em combinação, os riscos dessas toxinas sobre a comunidade zooplanctônica deveriam ser avaliados não apenas individualmente, mas também como misturas.